To determine the fate of the C-6 hydrogen of tetrahydrofolate in the ternary complex formed when Ch2-THG, thymidylate synthetase and FdUMP interact. Thymidylate synthetase is an excellent candidate for sophisticated physical studies by magnetic resonance and CD because 1) it is now available pure in large amounts by methods developed in this laboratory and the New England Enzyme Center 2) it involves a hydrogen transfer 3) it is a target for chemotherapeutic agents such as fluorinated pyrimidines and quinazoline derivatives 4) a partial reaction can be carried out with FdUMP as described in this application. We will study the incorporation of C13 and deuterated amino acids into the enzymes in vivo. Lactobacillus casei requires most of the common amino acids in its growth medium. The organism can be grown on a defined medium with the appropriate amino acid labeled. Tryptophan would be chosen first because there is evidence that it plays a role in the dihydrofolate reductase reaction as well. And its relation to niacin has led to speculation as to its possible role as a hydrogen carrier in enzymes (Schellenberg in "Pyridine Nucleotide Dependent Dehydrogenases" Springer Verlag, New York, 1970, p.15). We would initially test whether C14 labeled tryptophan is incorporated undiluted into the enzymes as expected. We would then isolate appropriately C13 labeled enzymes for NMR studies. Our laboratory will continue to evaluate new antifolates for their ability to inhibit microbial growth as well as folate requiring enzymes. Promising analogs are converted to their reduced forms and evaluated further as antitumor agents.